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Paracrine growth factor-mediated crosstalk between cardiac myocytes and non-myocytes in the heart is critical for programming adaptive cardiac hypertrophy in which myocyte size, capillary density, and the extracellular matrix function coordinately.
To examine the role that placental growth factor (PGF) plays in the heart as a paracrine regulator of cardiac adaptation to stress stimulation.
PGF is induced in the heart following pressure overload stimulation where it is expressed in both myocytes and non-myocytes. Here we generated cardiac-specific and adult inducible PGF overexpressing transgenic mice, as well as analyzed Pgf−/− mice to examine the role that this factor plays in cardiac disease and paracrine signaling. While PGF transgenic mice did not have a baseline phenotype or a change in capillary density, they did exhibit a greater cardiac hypertrophic response, a greater increase in capillary density, and increased fibroblast content in the heart in response to pressure overload stimulation. PGF transgenic mice showed a more adaptive type of cardiac growth that was protective against signs of failure with pressure overload and neuroendocrine stimulation. Antithetically, Pgf−/− mice rapidly succumbed to heart failure within a week of pressure overload, they showed an inability to upregulate angiogenesis, and they showed significantly less fibroblast activity in the heart. Mechanistically, we show that PGF does not have a direct effect on the cardiomyocytes but works through endothelial cells and fibroblasts by inducing capillary growth and fibroblasts proliferation, which secondarily support greater cardiac hypertrophy through intermediate paracrine growth factors such as interleukin-6.
PGF is a secreted factor that supports hypertrophy and cardiac function during pressure overload by affecting endothelial cells and fibroblasts that in turn stimulate and support the myocytes through additional paracrine factors.
Cardiac hypertrophy commonly occurs in response to a sustained elevation in workload, following an acute injury event or infection, or due to genetic mutations in genes encoding sarcomeric proteins. This process is partially adaptive through the increase in ventricular wall thickness that directly reduces wall stress and subsequent energy expenditure.1, 2 However, cardiac hypertrophy is also a risk factor for development of heart failure, arrhythmias and sudden cardiac death,3, 4 highlighting the need to differentiate between adaptive and maladaptive features of this process. Indeed, the myocardium can also hypertrophy in response to physiologic stimuli such as exercise and pregnancy without untoward effect.5
Although the growth of the cardiomyocyte is the most prominent feature associated with whole organ hypertrophy of the heart, it is now evident that this process also depends on the non-myocyte resident cells such as fibroblasts and endothelial cells, as well as changes in the extracellular matrix (ECM). Indeed, it is becoming clear that the ECM is a complex microenvironment containing a large cohort of differentially expressed proteins, signaling molecules, and barrier functions that play a major role in the hypertrophic response.6, 7 The growth of fibroblasts and ECM deposition, a process termed fibrosis, was considered to be a maladaptive response that contributes to ventricular arrhythmias, sudden cardiac death, and to left ventricular dysfunction.8 Yet, fibroblasts may also play an adaptive role in hypertrophy. For example, it was suggested that cardiac fibroblasts may support cardioprotection and cardiomyocyte hypertrophy, at least in part, by producing paracrine factors such as insulin-like growth factor I.9
Changes in capillary density also occur during hypertrophy. The number of capillaries increases in the early-compensated phase of hypertrophy in a mouse model of aortic constriction and then progressively decreases in mid to late stages of hypertrophy as the heart begins to decompensate.10, 11 This decline in capillary density leads to myocardial micro-ischemia that further exacerbates decompensation and leads to overt heart failure. Secreted proangiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietin-1 were shown to promote angiogenesis and prevent decompensation in this setting,11, 12 as well as other factors.10 Cardiomyocyte-specific deletion of VEGF-A in the mouse resulted in reduced capillary density and impaired contractility 12 and similarly, mice lacking angiopoietin-1 showed prominent myocardial defects with impaired vasculogenesis.13
Although it is clear that secreted growth factors and cytokines contribute to the non-myocyte response to hypertrophy, the identity of these remain elusive. Here we report on the identification of a secreted factor from the VEGF superfamily, placental growth factor (PGF), as an important mediator of the non-myocyte adaptive response to hypertrophy. PGF was previously shown to be increased in expression in the damaged human heart and under hypoxic conditions in cultured cardiomyocytes.14 Similarly, patients with ischemic cardiomyopathy or immediately after acute myocardial infarction show elevated PGF in their plasma.15, 16 Adenoviral-mediated PGF overexpression systemically in mice or direct injection of PGF protein into the rat heart each enhanced tissue perfusion or angiogenesis after myocardial infarction injury, suggesting a protective role for PGF.17, 18 Here we show that PGF serves as a paracrine support factor that directly influences non-myocytes in the heart, such as augmenting angiogenesis and fibroblast activity that then secondarily supports adaptive myocyte hypertrophy.
All procedures utilized in this study were described previously, although a full listing of the methods and materials is given in the online supplement.
PGF was identified as part of a larger project to classify novel secreted growth/paracrine factors produced in the heart by resident fibroblasts (data not shown). PGF mRNA expression was observed in isolated myocytes and non-myocytes (most of which were fibroblasts) from the adult heart, although expression was increased in myocytes from pressure loaded hearts induced by transverse aortic banding (TAC) (Figure 1A). Measurement of protein by ELISA from heart extracts showed that PGF is upregulated in the adult mouse heart following 2 or 7 days of pressure overload stimulation, but reduced to control levels by 14 days (Figure 1B). To investigate if PGF upregulation in myocytes was physiologically meaningful we generated transgenic mice to overexpress “low” levels of this factor in the adult heart using an inducible bi-transgenic system.19 A modified α-myosin heavy chain (α-MHC) promoter was used as the responder transgene to promote PGF expression when crossed with transgenic mice containing the α-MHC promoter-driven tetracycline transactivator (tTA) protein in the absence of Dox (Figure 1C). All mice were bred with Dox treatment to suppress transgene expression during embryonic and post-natal development, but removed at weaning (age of 4 weeks). Indeed, double transgenic mice (DTG) showed increased PGF expression by 4 weeks of Dox removal by both Western blotting and ELISA, which was increased to its maximum by 8 weeks off treatment (Figure 1D,E). To more rigorously quantify expression/secretion and interpret its physiologic significance in our DTG mouse hearts we analyzed PGF release from isolated myocytes and non-myocytes by ELISA . The data show that DTG myocytes released about 2-fold greater levels of PGF than myocytes from TAC stimulated hearts, or about the same level of PGF protein as resident non-myocytes from tTA control hearts (Figure 1F). Immunohistochemistry also showed extracellular PGF accumulation 8 weeks after Dox removal in DTG mouse hearts (Figure 1G). Importantly, the PGF transgene was not expressed outside myocytes in the heart (Supplement Figure I).
PGF DTG mice showed no overt cardiac phenotype at baseline as they aged to 8 months (6 months of effective overexpression). However, we hypothesized that PGF might only serve as an effector during pathophysiologic stimulation to function in concert with other secreted factors. Accordingly, PGF DTG and tTA control littermate mice were subjected to TAC beginning at 8 weeks of age (4 weeks after Dox removal) and assessed 2 weeks later. DTG mice showed significantly greater cardiac hypertrophy as measured by gravimetry and echocardiography (Figure 2A,B). Isolation of adult myocytes from these hearts showed an increase in myocyte length and width that was significantly greater in DTG mice compared with tTA controls (Figure 2C,D). Indeed, while total cellular area was increased to a larger extent in DTG myocytes, this was mainly due to an increase in width as shown by a decrease in the length/width ratio (Figure 2E,F). Importantly, ventricular performance was not negatively impacted by forced PGF expression in the heart after 2 or 12 weeks of TAC stimulation (Figure 2G,H), nor was baseline or dobutamine-stimulated function altered in PGF DTG mice as assessed with invasive hemodynamics (Supplemental Figure II). Thus, a physiological enhancement in PGF expression in the mouse heart induced significantly greater cardiac hypertrophy with TAC stimulation, but it did not lead to decompensation. In fact, PGF induction in the hearts of DTG mice was protective against a model of heart failure in which pressure overload stimulation was superimposed with neuroendocrine agonist infusion (necessary given the difficulty of inducing failure in the FVBN genetic background of mice). After 4 weeks of TAC stimulation combined with 1 week of angiotensin II/phenylephrine infusion, tTA control mice showed a significant reduction in cardiac function by echocardiography, which was better preserved in PGF DTG mice (Figure 2I). This result suggests that PGF expression/induction can be cardioprotective. There was no significant effect on induction of hypertrophic marker genes in the heart at baseline or after TAC stimulation with PGF overexpression versus control hearts (Supplemental Figure III).
PGF is a member of the VEGF family shown to promote angiogenesis at pharmacological doses. However, PGF DTG mice never showed an increase in myocardial capillary density in adulthood or with aging in the absence of a stimulus (Figure 3A, and data not shown). Therefore, we reasoned that PGF might require stimulation to have its pro-angiogenic effect in the heart. Indeed, 2 weeks of TAC stimulation induced significantly greater capillary formation in the hearts of PGF DTG mice compared with tTA controls (Figure 3A). Endothelial/hematopoietic progenitors (CD34) and total c-Kit containing progenitors (CD117) were also significantly increased in DTG hearts after TAC compared with control hearts (Figure 3B,C). In addition to an effect on endothelial cells and progenitor cells, increased PGF expression significantly augmented fibroblast number in DTG hearts after 3 days of TAC stimulation compared with tTA hearts, as assessed by immunohistochemical staining of vimentin (Figure 3D,E). This increase in fibroblast content was associated with significantly, albeit slight, increases in fibrosis with 2 weeks of TAC stimulation or aging in DTG hearts, as assessed by Masson’s trichrome histological staining or hydroxyproline biochemical analysis (Figure 3F,G). Taken together these studies show that PGF overexpression results in an adaptive remodeling of non-myocytes that mildly expands the ECM and capillary network in the heart, and even possibly the regenerative capacity of the heart.
The data presented above demonstrate a trophic role for PGF in facilitating growth of cardiac myocytes and expansion of endothelial cells and fibroblasts in the heart with disease-evoking stimulation. However, to ascertain the necessity or requirement of PGF in affecting these cell populations we analyzed mice deleted for the Pgf gene (Pgf−/−). Similar to PGF overexpressing DTG mice, Pgf−/− mice did not display a cardiac structural or functional deficit at baseline. We therefore subjected these mice and control Wt mice to pressure overload by TAC surgery. Unexpectedly, Pgf−/− mice showed higher mortality rates in response to this stress than Wt controls (data not shown). Therefore, we performed an analysis after only 1 week of TAC stimulation as fewer Pgf−/− perished in the first week. Gravimetric analysis showed an increase in ventricular weight normalized to body weight after 1 week of TAC in both Wt and Pgf−/− mice, although the increase was significantly greater in Pgf−/− mice (Figure 4A). However, echocardiographic assessment of Pgf−/− mice showed that their hearts were failing with a significant reduction in fractional shortening, impaired thickening of the ventricular walls and chamber dilatation (Figure 4B,C,D, and Supplemental Figure IV). In support of these observations, isolated adult cardiomyocytes from Pgf−/− hearts after 1 week of TAC showed a preferential increase in cellular length and a decrease in width, which is a hallmark of dilation and failure (Figure 4E,F). Indeed, while total area was increased more in isolated Pgf−/− cardiomyocytes, the length/width ratio was preferentially increased (Figure 4G,H), which is the opposite of the growth effect observed in PGF DTG myocytes from TAC stimulated hearts. Thus, while ventricular weights were increased in Pgf−/− mice after TAC stimulation, it was exclusively a non-adaptive type of dilatory growth that led to rapid failure. In addition, analysis of the hypertrophic markers showed increased expression of Nppa and Nppb in Pgf−/− mice after TAC versus controls, suggesting greater susceptibility to pathology (Supplemental Figure III).
PGF overexpressing mice displayed a higher angiogenic and fibrotic response following stress stimulation by pressure overload. To more definitively implicate PGF in these cellular effects we assessed these cell types in Pgf−/− mice after 1 week of TAC. Remarkably, Pgf−/− mice showed an inability to augment capillary density in the heart following 1 week of TAC, while Wt controls showed a robust response (Figure 5A). Pgf−/− hearts from TAC stimulated mice showed no difference in CD34 mRNA levels, although CD117 (c-Kit) was significantly reduced (Figure 5B,C). Masson’s trichrome histological staining did not show increased fibrosis at baseline or after 1 week of TAC in either genotype, as the fibrotic response is minimal at this time point. However, fibroblast numbers/proliferation was significantly reduced after 3 days of TAC stimulation in the hearts of Pgf−/− mice compared with Wt controls, as assessed by immunohistochemistry for vimentin (Figure 5D,E). These results are consistent with our observation that endogenous PGF is induced as early as 2 days after induction of pressure overload and suggest a role for PGF in the fibroblast and endothelial cell adaptive responses to pressure overload.
PGF is known to enhance VEGF activity on endothelial cells and promote angiogenesis.20 To evaluate this potential in a simulated cardiac milieu we performed co-culture experiments with neonatal rat ventricular myocytes infected with adenovirus (Ad) encoding PGF or β-galactosidase as a control. One day later, human umbilical vein endothelial cells (HUVECs) were plated on top the infected myocytes in Matrigel and then analyzed after another 24 hrs. A proangiogenic-like effect is quantified by scoring tube formation, and as predicted, AdPGF infected myocytes were significantly more effective than AdβGal infected cardiomyocytes in promoting tube formation (Figure 6A,B). AdPGF infection of primary fibroblasts showed a 20% increase in proliferation compared with control virus infection (Figure 6C), but it did not affect the growth of neonatal cardiomyocytes in culture after direct infection or with addition of recombinant protein (Figure 6E and data not shown). Consistent with these results and the likelihood that the PGF receptor is not expressed on cardiac myocytes, AdPGF infection only induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in non-myocytes from the heart (Figure 6D). Importantly, conditioned medium from AdPGF infected non-myocytes from the heart (mostly fibroblasts) induced a greater hypertrophic response in neonatal myocytes compared with medium from AdβGal infected fibroblasts (Figure 6E).
The results presented above suggest that PGF does not directly act on cardiac myocytes, though it still supports greater cardiac hypertrophy, which we hypothesized was due to secondary paracrine effectors released from fibroblasts and/or endothelial cells that were stimulated with PGF. Here we performed a selective qRT-PCR array for secreted growth factors from AdβGal or AdPGF infected cardiac fibroblasts (Figure 6F, Supplemental Table I). The data show a remarkable increase in expression of several growth factors by PGF in fibroblasts, many of which could easily support greater cardiac hypertrophy, such as IL6, IL1b, Cxcl1. Analysis of selected factors from this array in hearts of PGF DTG and Pgf−/− mice at baseline and after TAC stimulation showed that IL6, IL1b and Cxcl1 were dynamically regulated (Figure 6G,H and I). These results further define the potential mechanism of action for PGF in the heart, which supports adaptive cardiac hypertrophy by facilitating secondary growth factor release from non-myocytes in the heart.
The VEGF family includes VEGF, VEGF-B, placental growth factor (PGF), VEGF-C and VEGF-D. The deletion of VEGF specifically from cardiomyocytes in mice resulted in reduced capillary density and reduced contractility, suggesting that VEGF is important for the maintenance of cardiac function.12, 21 In a pressure overload model the repression of VEGF signaling with a decoy VEGF receptor resulted in contractile dysfunction and adverse cardiac remodeling, suggesting the importance of vascular growth during hypertrophy.22 Indeed, altered VEGF levels have been demonstrated in patients with heart failure, which correlates with the prognosis of these patients.23
PGF was originally discovered in the placenta, where it was proposed to control trophoblast growth, differentiation and vascular development.24, 25 PGF is a dimeric glycoprotein that, like VEGF, can bind with high affinity to the tyrosine kinase receptor vascular endothelial growth factor receptor 1 (VEGFR-1/Flt-1).26, 27 By comparison, VEGFR-2 (Flk1) is specific for VEGF and is not bound by PGF, and is the main effector of VEGF’s pro-angiogenic activity. PGF homodimers, presumably acting through VEGFR1, are chemotactic for cultured endothelial cells and monocytes and hence likely functions in concert with VEGF and the VEGFR2 in affecting angiogenesis.28 Importantly, the VEGFR1 (PGF receptor) is expressed more widely than just on endothelial cells and monocytes, such as on cardiac fibroblasts, and disputably cardiomyocytes.29, 30 Although both VEGF and PGF can bind VEGFR1, PGF homodimers uniquely stimulate the phosphorylation of specific VEGFR1 tyrosine residues and elicit a distinct response.20 Provocatively, cardiac overexpression of VEGF-B by AAV-mediated gene transfer in the rat produced ventricular hypertrophy that preserved cardiac function after infarction injury. 31 This result is interesting because like PGF, VEGF-B also only signals through VEGFR1.
Surprisingly, unlike VEGF, inactivation of the Pgf gene did not have an effect on vascular development in mice or otherwise impact their viability. However, deletion of PGF did retard pathological angiogenesis and collateral growth during ischemia.32 Conversely, systemic high dose production of PGF in a LAD ligation infarct model for 4 weeks stimulated angiogenesis in the infarct border and promoted vessel enlargement in the remote myocardium to improve cardiac recovery.17 Similarly, direct injection of PGF protein into the infarct region of the rat heart enhanced border zone angiogenesis, attenuated maladaptive ventricular remodeling, and preserved cardiac function.18 Here we showed that cardiac-specific PGF overexpression has no effect on the heart at baseline but it does improve the angiogenic response in mice subjected to pressure overload stimulation and preserve cardiac function better in response to a model of induced failure with TAC combined with neuroendocrine agonist infusion. By comparison, our studies in Pgf−/− mice show that endogenous PGF is required for adaptive angiogenesis and the prevention of heart failure in mice after pressure overload stimulation. We hypothesize that the inability to induce additional cardiac capillaries in Pgf−/− mice subjected to TAC likely underlies their susceptibility to heart failure. Indeed, deletion of Gata4 or hypoxia inducible factor-1 from the mouse heart prevented the adaptive augmentation in capillary density to pressure overload stimulation, which promoted decompensation and heart failure.11, 33
In addition to increased capillary density in PGF overexpressing mice and capillary rarefaction in the Pgf−/− mice, we also observed an enhanced fibrotic response with physiologic PGF overexpression. Interestingly, cardiac fibroblasts uniquely only express VEGFR1 suggesting that PGF may have a more specialized role in affecting this resident cardiac cell population versus VEGF, especially since PGF DTG mice show an increase in fibroblast content after TAC and mild fibrosis with normal aging. The accumulation of collagen and other ECM constituents is an integral feature of both pathologic and physiologic cardiac remodeling during hypertrophy.7 ECM proteins such as fibronectin, collagen and laminin directly bind to integrin receptors on the surface of cardiomyocytes to provide a mechanical or contact-based signaling that is constitutively sensed. For example, the deformation of the ECM during pressure overload activates integrins allowing the transduction of the mechanical force into intracellular pro-hypertrophic signaling.34, 35 Moreover, pathologic and physiologic cardiac remodeling changes the composition of the ECM through expression of other matricellular proteins such as periostin, osteopontin, thromobospondin-1/2, syndecan-1, and SPARC,36 which can affect myocyte growth characteristics through engagement of different receptors or integrins, or through binding and differential processing of secreted growth factors that bind these changing ECM components.
In addition to conditioning and remodeling the ECM during hypertrophy, the cardiac fibroblast has also been implicated in cardiac adaptation through paracrine effector secretion.6 In fact, several fibroblast-secreted factors have been demonstrated to support myocyte hypertrophy. For example, it was shown that the hypertrophic agent angiotensin II could induce fibroblast secretion of interleukin-6 (IL6) cytokine family members such as IL6, cardiotrophin-1 (CT-1), and leukemia inhibitory factor (LIF).37 These factors were shown to induce myocyte hypertrophy in culture or have been linked to cardioprotective effects in other systems.38 Here we used an array approach to show that PGF uniquely stimulated expression of many different growth factors in cardiac fibroblasts, many of which could explain the mild enhancement in cardiac hypertrophy through a paracrine effect on myocytes. Studies in vivo have shown that overexpression of periostin, a protein expressed exclusively by fibroblasts in actively remodeling hearts, can enhance hypertrophy.39 While PGF transgenic mice did show a mild but significant enhancement in cardiac fibrosis after TAC stimulation or with aging, they never decompensated even after 12 weeks of TAC stimulation and they were even protected from reductions in function using a novel model of heart failure induction (Figure 2I). PGF transgenic mice also demonstrated a small but significant enhancement in cardiac hypertrophy that was characterized by an adaptive pattern of myocyte growth in both length and width (preferentially in width though), while the heart failure observed in Pgf−/− mice after TAC stimulation showed myocyte lengthening with a loss of thickness, which is indicative of dilation and heart failure. Thus, we hypothesize that PGF is an adaptive mediator of ECM remodeling that supports compensated hypertrophy.
Our data show that PGF is an endogenous paracrine factor whose expression is predominantly induced in non-myocytes during hypertrophy. PGF stimulates the growth of capillaries and induces fibroblast proliferation to support hypertrophy and preserve cardiac function. Greater adaptive cardiac hypertrophy could be due to the release of secondary paracrine mediators from fibroblasts and endothelial cells to directly induce growth. ECM remodeling and controlled fibroblast activation might also alter the growth factor milieu associated with the accumulation of different matricellular proteins in the ECM, or these proteins might directly impact myocytes themselves through integrins and other receptors. Thus, PGF may serve as both a marker for adaptive cardiac remodeling and as a potential therapeutic tool in the future. However, caution should be noted since clinical trials in humans with ischemic heart disease using VEGF therapy has not shown efficacy in ischemic disease.40
This study was designed to examine the role that placental growth factor (PGF) plays in the heart as a paracrine regulator of cardiac adaptation to stress stimulation. We studied both PGF overexpression and deletion in pressure overload induced hypertrophy. Our results show the requirement of PGF for cardiac hypertrophy and compensation in response to pressure overload. In addition, we dissected the mechanism by which PGF confers cardioprotection and we show that PGF is a secreted factor that supports hypertrophy and cardiac function by affecting endothelial cells and fibroblasts that in turn stimulate and support the myocytes through additional paracrine factors.
Sources of funding This work was supported by grants from the National Institutes of Health (J.D.M.), the Fondation Leducq (Heart failure network grant to J.D.M), and the Howard Hughes Medical Institute (J.D.M.). F.C. was supported by a 1-year fellowship from the Italian Intesa SanPaolo SpA. P.C. was supported by the Flemish Government and Research Foundation Flanders (FWO) Research Project Funding.
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